Failure of survival of the embryo over the first weeks of its existence is considered to be a major cause of subfertility and infertility in mammals. This is particularly exemplified by embryos produced by assisted reproductive technologies (ART), including in vitro fertilisation (IVF) and all related techniques. A large proportion of ART embryos are lost during the pre- and immediate post-implantation periods through apoptosis.
A total of 27,067 ART treatment cycles were performed in Australia in 2000 (Hurst and Lancaster, 2001, AIHW National Perinatal Statistics Unit, Sydney, ISSN 10387234) resulting in 4,319 viable pregnancies (success rate of 16%). Given that an average of 2.1 embryos were transferred per treatment cycle (and over 90% of successful treatment cycles had 2 or more embryos transferred), this equates to less than 10% of embryos produced by ART having the capacity for long-term viability. ART is expensive. The average cost per treatment cycle in the USA is US$9547 (Collins, 2002, Human Reproduction Update. 8:265-77).
It is well established that much of the loss of embryo viability during ART occurs in the preimplantation phase or soon after implantation. ART causes a characteristic retardation of embryo development so that 96-120 h after fertilization embryos are commonly at least a full day behind their naturally produced counterparts in their developmental program. There are also fewer cells per embryo and many of the cells in embryos undergo apoptosis (Jurisicova et al., 1996, Molecular Human Reproduction. 2:93-8; O'Neill, 1997, Biology of Reproduction. 56, 229-237). In many cases the phenotype is sufficiently severe to result in the degeneration of the entire embryo. This retardation is a consequence of cellular stressors related to culture conditions. The preimplantation embryo constitutively expresses the machinery necessary for apoptosis and possesses the effectors and regulatory elements of apoptosis. Successful embryo development seems to require the suppression of this apoptotic machinery. Attempts at treatment of the various stressors have met with only limited success. For example, an interaction between oxidative damage and reduced stimulation of embryos by autocrine and paracrine growth/survival factors is a significant contributor to IVF-induced embryonic death. However relief from oxidative stress and provision of a wide range of putative embryonic survival/growth factors only partially ameliorate the effect of IVF. This suggests that there are other relevant stressors acting on the embryo, and/or that the nature of action of stressors on the embryo is not yet well defined.
Loss of embryo viability is a significant factor limiting the success of ART and there is a clear need to more completely elucidate the factors which lead to reduced embryo death during ART and to devise appropriate strategies to improve ART embryo viability.
Much is known about the response of somatic cells to various environmental stresses. For example, cells respond to many forms of genotoxic and nongenotoxic stress by the stabilisation and increased expression of the transcription factor p53 (see for example Agarwal et al., 1998, Journal of Biological Chemistry 273, 1-4). p53 is a ‘sensor’ of cell stress that plays an important role in maintaining normal genome stability. p53 operates within a complex network of interconnected cellular pathways by which cells sense and respond to inappropriate stresses. Other tumour suppressors operating within this network include, but are not limited to, Rb, PTEN, p21, p27, ARF and INK.
p53 has the capacity to either induce reduced cycle-cell progression (by the induction of CDK inhibitors such as p21) or to induce apoptosis (by inducing the synthesis of pro-apoptotic mediators such as Bax, PUMA, AlF, etc). Mutations in p53 lead to loss of regulation of cellular processes and are associated with the development of many cancers. Mutations in p53 are found in more than half of all human cancers. It is also now believed that many adult diseases derive, at least in part, from constraints during embryonic and fetal development, including during the embryo pre-implantation stage. Accordingly, an understanding of the stresses acting on the embryo and the embryo's response to these strategies will be important in devising strategies to minimise the onset of many adult diseases.
Preimplantation mammalian embryos normally produce an array of trophic factors that act to stimulate growth and survival of the embryo (Hardy and Spanos (2002) Journal of Endocrinology 172, 221-236). A major cause of the reduced viability of embryos produced by ART is diminished production of a number of these growth factors. For example, it has been observed that the production of platelet activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glyceryl-3-phoshocoline) and insulin-like growth factor II (IGF-II) is retarded in IVF-derived embryos (O'Neill et al., 1987, Fertility and Sterility 47, 969-975; and Stojanov et al., 1999, Molecular Human Reproduction 5, 116-124).
Such observations have led to the development of a range of protocols for the supplementation of in vitro embryo culture media with exogenous trophic factors, including PAF (Ryan et al., 1990, Journal of Reproduction and Fertility 89, 309-315; O'Neill et al., 1989, The Lancet ii, 769-772), in an effort to increase embryo viability. However the efficacy of such media supplementation is limited. Australian Patent No. 608530 describes the use of exogenous PAF or PAF analogue to increase the rate of implantation. However the effect observed was not a great as had been anticipated. Given the experimental evidence of the requirement for autocrine and paracrine trophic factors in normal embryonic development (O'Neill, 1997, Biology of Reproduction 56, 229-237), this clinical outcome is surprising.
Accordingly, there is a need for improved methods for enhancing embryo viability.